This patent application is a continuation-in-part from U.S. patent application Ser. No. 08/961,578 filed Oct. 31, 1997.
The present invention provides a novel phosphofructokinase isozyme (iPFK-2) that is preferentially transcribed and translated in tumor cells. The discovery of this isozyme, together with its function, led to the discovery of its use as a diagnostic target, as a drug screening target, and antisense compounds that inhibit its translation in cellular cytosol as an anti-tumor treatment.
The glycolytic pathway is a fundamental anaerobic pathway for sugar metabolism in eukaryotic cells. Glycolysis has a dual role, to degrade sugars to generate energy (ATP) and to provide building blocks for synthetic reactions. The rate of conversion of glucose into pyruvate is regulated to meet these two major cellular needs. In glycolysis, the enzymes hexokinase, phosphofructokinase and pyruvate kinase catalyze irreversible reactions and are regulated enzymes for control points in glycolysis. The enzymes are regulated by reversible binding of allosteric effectors, by covalent modification and by transcriptional control to meet changing metabolic needs. Of the three control enzymes, phosphofructokinase is the most important control point in mammalian glycolysis.
In 1930, Warburg pointed out that tumors have a high rate of anaerobic glycolysis and that they do not show a decreased glycolytic rate at relatively high O2 concentrations. This loss of regulatory control (i.e., the Pasteur effect) has come to be called the Warburg effect. Supplying tumor cells with glucose results in an inhibition of oxygen consumption, which magnifies the dependence on glucose for energy. Other cellular types do not normally show this effect since they maintain respiration from other substrates even in the presence of glucose. The question of why rapidly growing tumors have a marked tendency to convert the glycolytically-generated pyruvate to lactic acid in the cytosol instead of transporting into the mitochrondria for total oxidation has puzzled biochemists for years. The physiologic consequence of this altered metabolic behavior is clear. Tumor tissue generates a high degree metabolic inefficiency in the host, through an enhanced operation of energy-wasting processes, such as the Cori cycle between the tumor and the liver. As a result of the high glycolytic rate, a large amount of pyruvate is generated, together with an increase in the cytosolic NADH/NAD+ ratio, which favors the reduction of pyruvate to lactate through the action of lactate dehydrogenase. This is also supported by the low mitochondrial content of tumor cells which hampers the possibility of dissipating NADH through the action of the electron transfer chain and the low levels of NADH-shuttle systems found in a great number of tumors. The tumor cell becomes a lactate exporter in a similar way to some muscular fibers in anoxic situations. Although the precise role of the enhanced Cori cycle in a tumor-bearing states is not fully determined, it adds inefficiency to the host in a way that, instead of ATP formation of 36-38 molecules during the complete oxidation of glucose to CO2, a net loss of 4 ATPs can be expected when two three-carbon molecules are converted to one molecule of glucose.
A distinctive metabolic environment of cancer-bearing individuals has been described (Argilxc3xa9s and Azcxc3x3n-Bieto, Mol. Cell. Biochem. 81:3-17, 1988). Tumor invasion upon a host has been metabolically characterized by a reduction of the metabolic efficiency of the host, muscular protein depletion, increased gluconeogenesis, and uncoupling of oxidative phosphorylation. The net result is an energy imbalance leading to cachexia and eventual starvation.
The present invention provides a cancer malignancy diagnostic assay comprising obtaining a sample of a body fluid or tissue (including, for instance, a sample of tumor tissue, performing a sequence identity assay to look for the presence of iPFK-2 specific sequences (SEQ ID NO.: 11). Preferably, the sequence identity assay is selected from the group consisting of PCR (polymerase chain reaction) assays, ELISA immunologic assays, hybridization assays, and combinations thereof. The present invention further provides an anticancer, anti-inflammatory and cachexia pharmaceutical composition comprising a specific antisense oligonucleotide to the inventive isolated iPFK-2 sequence and a pharmaceutically acceptable oligonucleotide carrier. Preferably, the antisense oligonucleotide is a 15-50 base oligonucleotide incorporating an oligonucleotide sequence selected from the group consisting of: 5xe2x80x2-CCAACGGCATCTTCGCGGCT-3xe2x80x2 [SEQ ID NO: 2], 5xe2x80x2-GTCAGTTCCAACGGCATCTT-3xe2x80x2 [SEQ ID NO.: 4], and combinations thereof. The present invention further provides a therapeutic agent screening assay to screen for compounds having anti-tumor activity, comprising: (a) obtaining recombinant iPFK-2 having activity that forms fructose 2,6-bisphosphate from fructose 6-phosphate substrate; (b) adding candidate drug at various concentrations or no-drug control vehicle; and (c) assaying for fructose 2,6-bisphosphate as a measure of enzymatic activity. Preferably, the product assay is conducted by means of an enzymatic assay.
The present invention further provides a recombinant iPFK-2 polypeptide expressed by the cDNA sequence provided in SEQ ID NO. 11. The use of the iPFK-2 polypeptide, with known antibody techniques, including known monoclonal antibody techniques, further provides antibodies that specifically bind to iPFK-2. Preferably, such antibodies are monoclonal antibodies.